In the last decade, various methods have been developed to deliver genetic material into stem cells thr the specific control of gene expression. The most common methods include solution-based delivery using viruses, non-viral cationic lipids, nanoparticles, and polyplexes (see, e.g., Kim. C., et al., Mol. Pharm. 8, 1955-1961 (2011)). However, when treated with exogeneous materials, stem cells tend to die or undergo undesired differentiation patterns. Therefore, there are concerns associated with the introduction of viruses, nanoparticles, and other exogenous materials into stem cells (Yoo, J. W., et al., Nat Rev. Drug Discov. 10, 521-535 (2011)). Another commonly used technique, which circumvents this issue, is electroporation (Hoelters, J., et al. J. Gene Med. 7, 718-728 (2005)). However, electroporation has been shown to cause high levels of cell death due to physical damage to the cell membrane in addition to the introduction of other undesired materials into the cell (e.g. ions, which can shill the concentration gradient) (Beebe, S. J., et al., Faseb J. 17, 1493-1495 (2003)). Therefore, the development of new methods to safely and effectively deliver genetic materials into stem cells is needed.
Recently, increasing attention has been given to substrate-mediated genetic delivery, in which cells uptake biomaterials from the substrate on which they are grown. These methods can potentially facilitate the uptake of genetic material in a noninvasive fashion and prevent the need to deliver exogenous materials. Shalek et al. reported that silicon nanowires, which physically impale cells, can deliver genes and siRNA (Shalek, A. K., et al., Proc. Natl. Acad. Sci. USA 107, 1870-1875 (2010)). This system has been shown to be very efficient at delivering genetic material into a variety of cell types, including stem cells. Nevertheless, the mechanism governing how nanowires affect cell physiology remains to be investigated. Reverse transfection, or the substrate-mediated uptake, of siRNA by cells has also been explored using the layer-by-layer technique (Zhang, X., et al., Biomaterials 31, 6013-6018 (2010)). However, such techniques require the use of cationic polymers, similar to the ones used in solution-based transfection, which may not be ideal in terms of maintaining stem cell viability. Therefore, there is a pressing need to further develop and characterize substrate-mediated strategies that can facilitate nucleic acid delivery into stem cells. These techniques are of particular importance for investigating and controlling differentiation.
The stem cell microenvironment plays a major role in controlling various stem cell behaviors. It has already been demonstrated that stem cell fate can be controlled by making ECM protein patterns of different geometries and dimensions (Solanki, A., et al., Small 6, 2509-2513 (2010); Guilak, F., et al., Cell Stem Cell 5, 17-26 (2009)). While stem cell differentiation can be controlled by manipulating the expression of certain genes, it remains a question whether topographical features of the ECM can be utilized to control this expression.
One of the critical barriers to harnessing the full therapeutic potential of stem cells is the development of an easy, effective, and non-toxic methodology to control differentiation into specific cell lineages. Stem cell differentiation can be controlled by modulating key gene expression levels or signaling pathways within the cell, which has been achieved by several conventional gene delivery methods. For example, the RNA interference (RNAi) method. For controlling gene expression levels using siRNA or miRNA is emerging as an important tool in stem cell biology. For the successful genetic manipulation of stem cells, the cells must typically maintain their viability for an extended period of time after single or multiple siRNA transfections, without affecting the intrinsic cellular functions. However, many of the conventional methods used to deliver siRNA into stem cells, including lipid-based transfections, viral vectors, nanowire-based platforms, and electroporation techniques, result in significant cytotoxicity and undesirable side-effects. This presents a considerable challenge for the development of both, robust and reliable siRNA delivery into stem cells to control their differentiation into the desired cell lineages.
Currently, one of the most common methods to deliver siRNA into stem cells is the solution-mediated delivery (or forward transfection) using exogenous chemical materials including non-viral cationic lipids, nanoparticles, and polymers. However, such exogenous materials may be cytotoxic for the delivery of siRNA into stem cells and thereby need to be removed after a certain incubation period. In addition, they can potentially compromise the ability of stem cells to proliferate, migrate and differentiate. Therefore, there are several limitations associated with the solution-mediated delivery methods for manipulating gene expression within stem cells. In order to address these limitations, increasing attention has been given to the substrate-mediated delivery of siRNA, wherein the cells directly uptake the siRNA from the underlying substrate. Substrate-mediated delivery can potentially facilitate the uptake of siRNA into stem cells, which precludes the need to use exogenous materials as delivery vehicles. For instance, it was reported that silicon nanowires, which physically impale the cell membrane, can deliver siRNA into the cellular cytoplasm. Nevertheless, the potential physical damage caused by the nanowires on the plasma membranes of cells and the mechanism of how the nanowire arrays transfer siRNA into cells was not addressed. Moreover, the survival of stem cells for extended periods, which is required for their differentiation, was not demonstrated. Thus, there is a clear need to develop nontoxic, and efficient strategies to deliver siRNA into stem cells to control gene expression levels, such that we can maintain the biological functions of stem cells for extended periods of time and efficiently control their differentiation into specific cell types.